The goal of this proposal is to examine the structural details of two key receptor/ligand interactions that facilitate the transport of antigens and antibodies across the mucosal epithelium, a constraint that must be overcome in order to effectively mount immune responses in the mucosal compartment by: 1) determining the three-dimensional structure of Neutrophil Gelatinase Associated Lipocalin; 2) characterizing the interaction between the Polymeric Immunoglobulin Receptor and its ligand/s through directed mutagenesis and surface plasmon resonance- based binding assays; and 3) crystallizing the extracellular moiety of the human Polymeric Immunoglobulin Receptor. (1) Neutrophil Gelatinase Associated Lipocalin (NGAL) is a member of the lipocalin family of binding proteins. Expressed by neutrophils and induced in epithelial cells in both inflammatory and neoplastic colorectal disease, NGAL binds gelatinase and N-formylated peptides. NGAL plays an important role in mediating epithelial inflammatory responses and has been implicated in tumorigenesis. We have determined crystallization conditions for human NGAL and have collected high-resolution diffraction data from cryo- preserved crystals of NGAL plus/minus ligand. (2) Mucosal epithelium comprises the linings of the gastrointestinal, respiratory and urogenital tracts and constitutes a major route for infection by pathogens. Multimeric immunoglobulin A (IgA) is the predominant antibody in the mucosa and is transported there, across the epithelium, through a specific interaction with the polymeric immunoglobulin receptor (pIgR). We propose to examine the structural details of this interaction through a combination of approaches. We have developed baculovirus-based expression systems for a functional form of the extracellular domain of the human pIgR (Secretory Component; SC) as well as J chain, a component of polymeric antibodies (IgA and IgM). Using surface plasmon resonance-based binding assays, we will study the interaction between our expressed SC and: polymeric antibodies from various sources; our expressed J chain; and a series of proposed J chain mutants. (3) We have determined preliminary crystallization conditions for our expressed SC. The resources currently available are insufficient to produce enough protein to fully characterize the crystallization conditions or support a subsequent crystallographic analysis.